CN109486956B - Multi-group integrated precise breeding method for pigs - Google Patents
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Abstract
The invention discloses an SNP locus related to pig farrowing traits, which is positioned at 323407415 th site of pig HSD17B1 gene, wherein two allelic genes of the SNP locus are A or G; the three genotypes corresponding to the SNP sites are AA, GG and AG respectively, and compared with the swine with the genotype of AA, the swine with the genotype of AG and GG has higher litter size and litter weight. On the basis of the SNP loci, the genetic progress of the pigs with high production efficiency can be accelerated by selecting the genotypes.
Description
Technical Field
The invention relates to a multi-group chemical integration accurate breeding method for pigs, in particular to a multi-group chemical integration accurate breeding method for improving the breeding efficiency of pigs.
Background
The pig raising industry in China has a long history, and the pig breeds have rich resources, wide distribution, huge utilization potential and wide development prospect. The production of the breeding pigs is the basis of the production of the pig industry, and the breeding pigs can breed a large number of piglets with high quality only through scientific feeding management, so that the economic benefit of the pig industry can be improved.
With the continuous and rapid development of economy in China, the living standard of people is gradually improved, and the demand of pork is higher and higher, so people pay more and more attention to how to improve the reproductive performance of pigs. Reproductive performance is an important economic trait of pigs, including litter size, birth weight and litter weight, weaning litter weight, day age of birth, litter interval, and the like. The breeding of the reproductive capacity of the sow plays an important role in improving the overall benefit of pig raising production, brings great economic benefit to the pig raising industry, and is one of important factors influencing the production efficiency of the pig raising industry.
On the one hand, current methods to obtain high reproductive efficiency pigs focus mainly on genome editing. The genome editing technology developed on the basis of the transgenic technology and the somatic cell cloning technology can realize accurate modification of the genome by editing target genes, including knocking out specific DNA fragments, introducing specific mutations, transferring the specific DNA fragments and the like. China has rapid research progress in relevant aspects of genome editing pigs, and obtains a series of agricultural and medical engineering pigs. Researchers introduce beneficial genes into specific sites through gene operation, so that the stability and efficiency of exogenous genes are improved, the genetic specificity of animals is changed, such as the production performance of the animals is improved, and finally, new high-yield, high-quality and disease-resistant animal varieties meeting the requirements of people are bred. In addition, some genes for negatively regulating the growth of the animals can be modified or knocked out, and high-yield transgenic animals can be cultured.
However, people increasingly pay attention to the effect of pig breeding methods on pig quality and the influence on human health while the requirement on pork yield is improved. Because the scientific community is difficult to prove that transgenic animals have no side effect on the health of human beings in a short time and the cognition deficiency of the broad masses on the transgenic animals, people have strong desire for eating the non-transgenic animals, and the price of the non-transgenic animals is higher than that of the transgenic animals.
On the other hand, apart from genome editing techniques, the realization of other breeding theories also requires an understanding of the pig genome. However, the understanding and recognition of pig genome is far from sufficient, and the control of genes affecting pig breeding efficiency (including pig litter size, average litter weight, etc.) is not yet complete, and further research is necessary to meet the needs of pig breeding methods.
Therefore, a brand-new high-breeding-efficiency pig breeding method needs to be established urgently to meet market demands, improve economic benefits, realize the promotion of pig breeding theories and technical levels in China and realize the cross-over development of pig breeding in China.
Disclosure of Invention
The inventor carries out intensive research, enriches SNP sites influencing the pig breeding efficiency by a whole genome association analysis method aiming at the problems in the pig breeding method, and also provides a multigroup chemical integration accurate breeding method for improving the pig breeding efficiency.
The object of the present invention is to provide the following:
(1) an SNP site related to the piglet litter size trait, wherein the SNP site is located at 323407415 th site of porcine HSD17B1 gene, and two alleles of the SNP site are A or G.
(2) The SNP site according to the item (1), wherein the three genotypes corresponding to the SNP sites are AA, GG and AG, and the pigs with the genotypes of AG and GG have higher litter size and litter weight compared with the pigs with the genotypes of AA.
(3) The SNP site according to (1) above, wherein the method for detecting the SNP site comprises a set of amplification primers P1And P2The amplification primer P1And P2The nucleotide sequences of (A) and (B) respectively comprise sequences shown in SEQ ID NO.1 and SEQ ID NO. 2.
(4) The SNP site according to (1) above, wherein the method for detecting the SNP site comprises extension primers, and the nucleotide sequences of the extension primers respectively comprise the sequences shown by SEQ ID NO. 3.
(5) The SNP site according to (1) above, wherein the kit for detecting the SNP site comprises a set of amplification primers P1And P2The amplification primer P1And P2The nucleotide sequences of (A) respectively comprise sequences shown in SEQ ID NO.1 and SEQ ID NO. 2; and/or
The kit for detecting the SNP locus comprises extension primers, wherein the nucleotide sequences of the extension primers respectively comprise sequences shown in SEQ ID NO. 3;
specifically, the kit for detecting the SNP locus comprises a PCR reaction reagent, a PCR amplification primer, a PCR product purification reagent, an extension primer and an SNP locus control specimen.
(6) A method for identifying or assisting in identifying the litter size traits of pigs comprises the steps of detecting whether the 323407415 th deoxyribonucleotide of a porcine HSD17B1 gene is A, or G, or A and G to determine the genotype of a pig to be detected is AA, GG or AG, or directly determining the 323407415 th genotype of the HSD17B1 gene by a method such as Sequenom MassArray and the like, and determining the litter size and/or litter weight of the pig according to the genotypes: pigs of genotype AG and GG had higher litter size and/or litter weight compared to pigs of genotype AA,
wherein, the AA genotype is a homozygote of the 323407415-bit deoxyribonucleotide of the porcine HSD17B1 gene, namely A; GG genotype is a homozygote of the 323407415-bit deoxyribonucleotide of the porcine HSD17B1 gene, namely G; the AG genotype is a heterozygote of 323407415-position deoxyribonucleotide of a porcine HSD17B1 gene, A and G.
(7) A breeding method of high-yield litter size/weight pigs, wherein the breeding method comprises the step of selecting pigs with GG or AG genotype, preferably GG genotype at position 323407415 of HSD17B1 gene as parents for hybridization.
(8) A breeding method according to (7) above, wherein the breeding method comprises the steps of:
step 1, selecting a pig with the 323407415 th site of HSD17B1 gene being GG or AG genotype as a parent to hybridize to obtain an F1 generation piglet;
step 3, carrying out genotype analysis on the F2 generation piglets, breeding piglets with GG or AG genotypes at the 323407415 th site of the HSD17B1 gene, and continuously hybridizing after the piglets grow up to obtain F3 generation piglets;
and (3) repeating the step 2 or the step 3 to finally obtain the breeding pig with the genotype GG high inheritance.
(9) The use of the SNP site described in (1) above in identification or assisted identification of swine litter traits and breeding.
(10) The use of the amplification primer for detecting the SNP site in (3) above in identification or auxiliary identification of pig litter traits and breeding; and/or
The use of the extension primer for detecting the SNP site in (4) above in identification or auxiliary identification of swine litter traits and breeding.
The swine polyculture integration accurate breeding method provided by the invention has the following beneficial effects:
1. the SNP locus related to the litter size traits of the pigs, which is discovered by the invention, can be used for promoting the breeding selection of high-yield litter size pigs and accelerating the breeding process;
2. the invention provides a kit for detecting SNP loci and genotyping, which is convenient to use, simple to operate and low in cost;
3. the selected SNP locus, amplification primer or extension primer related to the litter size traits of the pigs can be effectively used for identifying or assisting in identifying the pigs with high litter size/litter weight and accelerating the genetic progress of the pigs with high litter size.
Drawings
FIG. 1 is a graph showing the results of HSD17B1 gene expression levels of Meishan pigs and big white pigs in example 2.
Detailed Description
The features and advantages of the present invention will become more apparent and appreciated from the following detailed description of the invention. The word "exemplary" is used exclusively herein to mean "serving as an example, embodiment, or illustration. Any embodiment described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments.
The invention aims to overcome the defects of the prior art and provide an SNP locus related to the pig production efficiency, namely the litter traits and the identification and application of the SNP locus. The SNP loci related to the piglet traits are searched by using a whole genome association analysis method, and the SNP loci related to the piglet traits are applied to the selective breeding of pig breeds.
In the invention, a large white pig or a Meishan pig is selected to identify SNP sites related to litter traits. The white pig and the Meishan pig account for higher proportion in the breeding of the pig breeds in China, and the two pigs are selected to facilitate the subsequent breeding of the pig breeds in China.
The invention uses a whole genome association analysis method to search SNP sites related to the pig litter traits. The global genome association analysis (GWAS) is a method for scanning a population to be researched by screening high-density molecular markers in a global genome range, analyzing and scanning the association relation between molecular marker data and phenotypic traits, and is also the most widely used method for screening related SNP sites at present. However, the method of whole genome association analysis is used for searching SNP sites related to the pig litter traits, the obtained results are numerous and disordered, and most of gene variation is not related to the target traits. It is quite difficult to select the relevant SNP site in the result of this bulky disorder. Therefore, it is necessary to select a specific gene in advance and determine the relevant SNP site located on the specific gene in combination with the GWAS method.
The invention selects specific genes from a plurality of screened genes to further determine the SNP sites on the genes, thereby greatly improving the success rate of determining the related SNP sites.
In a preferred embodiment, the specific gene is HSD17B1 gene.
The HSD17B1 gene is a member of HSD17B family, mainly plays a role in the last step of estrogen biosynthesis, has an important regulation and control role in the processes of animal growth, development and reproduction, and can convert estrogen into estradiol with higher bioactivity. At present, the HSD17B1 is associated with the occurrence and the development of certain reproductive diseases, but no clear conclusion is made. The expression of HSD17B1 gene is detected by researchers in both breast cancer and endometrial cancer; the correlation between the polymorphism or variation of the HSD17B1 gene and the breast cancer and the endometrial cancer is also researched by different researchers respectively, and the result shows that the polymorphism or variation of the HSD17B1 gene is not significantly correlated with the researched cancer.
Meanwhile, the pig production efficiency is known to comprise productivity indexes such as litter size, litter weight and litter weight. The litter size can be directly related to the quantity of the fattening pigs and the pork supply quantity; the average birth weight is large, the variation coefficient is small, the litter regularity is good, the survival rate of piglets is improved, and the average birth weight is large and the sow can be stimulated to produce more milk; the litter weight is a comprehensive consideration of the litter size and the tire weight. The inventors believe that the above-mentioned indicators are all related to litter trait candidate genes: fetal survival rate has an important relationship with fetal weight of pigs, the relative size of the placenta is one of the main factors affecting fetal survival rate, and the chorioepithelia and the endometrial epithelia form folds during gestation of piglets, and the fold folding level affects the relative size of the placenta. Therefore, the inventor considers that the HSD17B1 gene indirectly related to the size of the pig milk production and the placenta is a candidate gene of farrowing traits influencing the production efficiency of the pig.
At present, no report about the association of HSD17B1 gene and pig litter size trait exists. The invention uses a whole genome association analysis method to carry out association analysis on the polymorphism of the gene and important reproduction traits, and determines the SNP site of the litter trait in the pig gene for the first time through a large amount of researches.
By combining a GWAS method with a screened specific gene, the invention obtains/provides an SNP locus related to pig production efficiency, namely litter traits, wherein the SNP locus is located at 323407415 (rs323407415) of HSD17B1 gene, and two alleles of the SNP locus are A or G. Preferably, the gene frequency of nucleotide A is 0.159 and the gene frequency of nucleotide G is 0.841.
Three genotypes corresponding to the SNP locus are AA, GG and AG respectively, wherein the AA genotype is a homozygote of the 323407415 th deoxyribonucleotide of a porcine HSD17B1 gene which is A; GG genotype is a homozygote of the 323407415-bit deoxyribonucleotide of the porcine HSD17B1 gene, namely G; the AG genotype is a heterozygote of 323407415-position deoxyribonucleotide of a porcine HSD17B1 gene, A and G.
The present inventors have conducted extensive studies to find that pigs with genotypes AG and GG have higher litter size and litter weight compared to pigs with genotypes AA.
In the invention, a Sequenom MassArray detection system is used for determining the genotyping of the SNP locus. The basic principle of the Sequenom MassArray detection system is a matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) technology, a target sequence is firstly amplified by PCR, then an SNP sequence specific extension primer is added, and 1 base is extended on an SNP site. The prepared sample analytes were co-crystallized with the chip matrix and subjected to transient nanosecond (10) in a vacuum tube of a mass spectrometer-9s) strong laser excitation, nucleic acid molecule desorption and conversion into metastable state ion (mostly single charged ion), ion flight time in the power plant is inversely proportional to ion mass, and the flight time of nucleic acid molecule in the vacuum tube is detected by the flight time detector to obtain the accurate molecular weight of sample analyte, thereby detecting SNP site information.
As described above, the SNP site genotyping measurement involves steps such as extraction of DNA from pig genome, PCR amplification reaction, and single base extension reaction.
In a preferred embodiment, the amplification primer in the PCR amplification reactionP1And P2The nucleotide sequences of (A) are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2.
In a preferred embodiment, the nucleotide sequence of the extended primer in the single base extension reaction is shown as SEQ ID NO. 3.
The invention provides a detection method for SNP locus genotyping related to pig litter traits, which comprises the following steps:
(1) extracting the genomic DNA of the pig;
(2) and (3) taking the genomic DNA of the pig to be detected as a template, and performing Sequenom MassArray detection by using an amplification primer and an extension primer to determine the genotype of 323407415 th site of the pig HSD17B1 gene.
Wherein the amplification primer (P) in the PCR amplification reaction1And P2) As shown in SEQ ID NO.1 and SEQ ID NO. 2; the extension primer is shown as SEQ ID NO. 3.
Based on the SNP locus and the genotyping related to the litter traits, the invention provides a method for detecting the SNP locus related to the litter traits of pigs, and the detection method comprises direct sequencing or kit determination.
The kit comprises (1) a PCR extension primer of an SNP locus; preferably, the kit also comprises (2) PCR reaction reagents: comprises PCR buffer solution, dNTP, PfuDNA polymerase and SNaPshot mixed solution; (3) PCR amplification primer P1And P2(ii) a (4) PCR product purification reagents: comprises exonuclease, shrimp-alkaline phosphatase and buffer solution for purification; (5) SNP site control specimen: comprises a homozygous mutation positive control specimen, a heterozygous mutation positive control specimen and a negative control specimen.
The SNP homozygous mutation positive control specimen is a plasmid which contains G basic groups artificially synthesized by a pig rs323407415 locus.
The SNP heterozygous mutation positive control specimen is a mixture of two plasmids containing artificially synthesized A and G basic groups at the site rs323407415 of a pig.
The SNP negative control specimen is an artificially synthesized plasmid with random bases. The quality control of the reaction system by using the three control samples can ensure the consistency of the detection result.
The use method of the detection kit provided by the invention comprises the following steps: extracting a DNA sample from pig ear or other tissue cells; preparing a PCR reaction system, carrying out PCR amplification and purifying a PCR product; simultaneously carrying out extension reaction on the PCR product and the extension primer of the SNP locus; performing capillary electrophoresis analysis on the extension product; SNP locus analysis, and further gene typing information can be obtained. The SNP site analysis can adopt, but is not limited to, an ABI3730XL automatic sequencer.
Further, the invention provides a method for identifying or assisting in identifying the litter size traits of pigs, which comprises the steps of detecting that the 323407415-position deoxyribonucleotide of the HSD17B1 gene of the pigs is A, G or A and G, determining that the genotype of the pigs to be detected is AA, GG or AG, or directly determining the genotype by methods such as Sequenom MassArray and the like, and determining the litter size and/or litter weight of the pigs according to the genotype: pigs of genotype AG and GG had higher litter size and/or litter weight compared to pigs of genotype AA,
wherein, the AA genotype is a homozygote of the 323407415-bit deoxyribonucleotide of the porcine HSD17B1 gene, namely A; GG genotype is a homozygote of the 323407415-bit deoxyribonucleotide of the porcine HSD17B1 gene, namely G; the AG genotype is a heterozygote of 323407415-position deoxyribonucleotide of a porcine HSD17B1 gene, A and G.
Furthermore, the invention provides a breeding method of pigs with high litter size/litter weight, which comprises the step of selecting pigs with GG or AG genotype, preferably GG genotype at position 323407415 of HSD17B1 gene as parents for hybridization. Specifically, the method comprises the following steps:
step 1, selecting a pig with the 323407415 th site of HSD17B1 gene being GG or AG genotype as a parent (F0) to hybridize to obtain a first generation (F1 generation) piglet;
and 3, carrying out genotype analysis on the F2 generation piglets, breeding piglets with GG or AG genotypes at the 323407415 th site of the HSD17B1 gene, and continuously hybridizing after the piglets grow up to obtain the F3 generation piglets.
And (3) repeating the step 2 or the step 3 to finally obtain the breeding pig with the genotype GG high inheritance. The method follows natural propagation and obtains the breeding pigs with high litter size/weight through less manual intervention; meanwhile, the breeding method is a non-transgenic method, the acceptance of the masses is higher, the development of the pig industry can be effectively promoted, and the core competitiveness of the pig breeding industry in China is improved.
Furthermore, the invention also provides the application of the method in the aspects of identifying and/or breeding the pig breeds based on the above disclosure according to the SNP sites, the extension primers, the amplification primers, the SNP site testing method, the SNP site detection kit and the SNP site genotyping, wherein the pig breeds have higher production efficiency, and the production efficiency comprises litter size and litter weight.
The inventor considers that: the effect of DNA level variation on the phenotypic difference of pig groups and individuals is overestimated, the genome expression regulation mechanism is not completely clear, the effect of multilayer omics on the phenotype is underestimated, and the personalized genetic mechanism of the pig species is not concerned. The huge phenotypic genetic difference between pig breeds and individuals depends not only on the variation of the genome structure and the DNA sequence, but also on the multi-level expression regulation of the genome. In order to realize the efficient application of genome information, it is necessary to develop research on multiclass variation of pigs, integrate multiclass information, establish a population personalized multiclass integrated precise breeding theory and technical system, and realize multiclass selection. The innovation and the application of multigroup theory of integrated precise breeding are realized, and the pig breeding theory and the technical level in China are obviously improved.
Based on the consideration, the invention analyzes and verifies the factors influencing the pig production efficiency on the transcription level.
The invention provides a gene related to pig production efficiency, namely farrowing traits, wherein the gene is a pig HSD17B1 gene.
Furthermore, the invention provides a method for identifying or assisting in identifying the farrowing traits of the pigs, which determines the farrowing traits of the pigs by measuring the expression level of the mRNA of the HSD17B1 gene of the pigs.
The method for identifying or assisting in identifying the litter traits of the pigs comprises the steps of obtaining total RNA of ovarian tissues or other tissue cells of the pigs, and determining the expression quantity of mRNA of HSD17B1 gene of the pigs through reverse transcription and quantitative PCR, wherein the lower the expression quantity of the mRNA, the higher the litter size and/or litter weight of the pigs, and the higher the expression quantity of the mRNA, the lower the litter size and/or litter weight of the pigs.
Wherein, the quantitative PCR amplification procedure comprises the following steps: pre-denaturation at 95 deg.C for 5 min; denaturation at 95 ℃ for 5 s; extending at 60 ℃ for 30 s; repeat 40 cycles; 95 ℃ for 15 s; 60 ℃ for 1 min; 95 ℃ for 15 s; 60 ℃ for 15 s.
The invention provides a PCR amplification primer P for determining the expression quantity of HSD17B1 gene mRNA3And P4，P3And P4The base sequences of (A) are respectively shown as SEQ ID NO.4 and SEQ ID NO. 5.
The invention provides a kit for determining the expression quantity of mRNA of HSD17B1 gene, which comprises an amplification primer P3And P4，P3And P4The base sequences of (A) are respectively shown as SEQ ID NO.4 and SEQ ID NO. 5.
Further, the present invention provides a breeding method of high litter size pigs, comprising the step of selecting a pig with low expression level of HSD17B1 gene mRNA as a parent for hybridization. Specifically, the method comprises the following steps:
step 1, selecting a pig with low expression quantity of HSD17B1 gene mRNA as a parent (F0) to hybridize to obtain a first generation (F1 generation) piglet;
and 3, carrying out mRNA quantitative analysis on the F2 generation piglets, breeding piglets with low expression quantity of HSD17B1 gene mRNA, and continuously hybridizing after the piglets grow up to obtain the F3 generation piglets.
Repeating the step 2 or 3 to finally obtain the low-expression HSD17B1 and the high-inheritance boar.
The invention also provides application of the HSD17B1 gene, the amplification primer, the expression quantity test method and the expression quantity detection kit based on the disclosure in the aspect of identifying and/or breeding the boar according to the HSD17B1 gene, the amplification primer, the expression quantity test method and the expression quantity detection kit related to the farrowing characters of the boar, wherein the boar has higher production efficiency, such as higher litter size.
Combining the analysis of the gene level and the analysis of the transcription level, and using a multidimensional metamics technical means system to illustrate the effect of genetic variation on selection and domestication, the multidimensional metamics selection of a target group is realized, the accuracy of pig breeding is improved, and the pigs with high production efficiency can be accurately obtained.
Examples
EXAMPLE 1 obtaining molecular markers for SNPs
1. Experimental sample Collection
375 big white pigs from certain pig farms in Shandong were used for SNP typing of HSD17B1 gene. Ear tissue samples were collected one by one, placed in 75% alcohol and stored at-20 ℃ for DNA extraction.
2. SNP typing of HSD17B1 Gene
2.1 extraction and detection of pig genomic DNA
(1) Appropriate amount of pig ear tissue was cut, minced and placed into 1.5mL Axgen tubes.
(2) A50 mLBD centrifuge tube was used, proteinase K at a final concentration of 0.4mg/mL and lysis buffer were mixed well, and 0.5mL of lysis buffer was added to a 1.5mL centrifuge tube containing porcine ear tissue for lysis.
(3) The centrifuge tubes were placed in parallel and uniformly on a rocking plate of a constant temperature hybridization oven (the tube caps were tightly sealed to prevent liquid from leaking out). Standing at 55 deg.C for more than 6 hr (it is important to mix the sample thoroughly during digestion process, the judgment is based on no obvious macroscopic pig ear tissue, and the digested mixture is milky);
(4) after the sample is fully cracked, taking out the centrifuge tube, adding 0.3mL of saturated sodium chloride solution into each tube, reversing and fully mixing for 6-8 times, then placing on ice, and carrying out ice bath for 15 minutes;
(5) after ice-cooling, centrifuge at 12000rpm for 15 minutes at room temperature, carefully and slowly transfer the supernatant to a new 1.5ml axgen centrifuge tube (care was taken to avoid the pellet from being poured out with the supernatant, keeping the pouring procedure consistent, and keeping the amount of supernatant poured out of each tube the same);
(6) adding 0.7mL of isopropanol (the amount of isopropanol added varies with the amount of supernatant poured off, and the two are equal in volume) to each tube, and inverting until flocculent precipitate appears in the solution (if there is no flocculent precipitate, the solution can be left in a refrigerator at-20 ℃ for 2 hours or at 4 ℃ overnight);
(7) centrifugation at 12000rpm for 15 minutes at room temperature removed the supernatant (during which time the tube was carefully observed for white DNA pellet at the bottom of the tube and was not decanted with the supernatant);
(8) 0.5mL of 70% ethanol was added to each tube and inverted gently to wash the precipitated DNA thoroughly;
(9) centrifugation at 10000rmp for 30s, and aspiration of ethanol from the centrifuge tube with a 200. mu.L micropipette to leave the precipitated DNA in the tube (when one is not concerned with aspirating the DNA precipitate from the tube, the tip used in this step may not be replaced when operating between centrifuge tubes);
(10) naturally air-drying the DNA for 10 minutes;
(11) taking 0.1mL of TE buffer solution by a pipette, dissolving the DNA precipitate again, placing the DNA precipitate at 55 ℃ for 2 hours, and shaking for several times to fully dissolve the DNA;
(12) after the DNA is fully dissolved, measuring the extracted concentration by using an ultraviolet spectrophotometer (genotyping needs to standardize the DNA concentration and OD value of a sample, the DNA concentration is 15-20 ng/mu L, the volume is 30 mu L, A260/230 is between 1.5 and 2.3), and detecting the extracted mass (a single band can be seen) by agarose gel electrophoresis.
(13) The DNA solution was left at 4 ℃ overnight, and 1. mu.L of the DNA solution was subjected to PCR the next day (if the extracted DNA was used within several weeks, it could be stored at 4 ℃ C.; if it was not used for a long time, it was left at-20 ℃ C.).
2.2 genotype determination and quality control of genotype data for SNP chip
DNA samples of 375 large white pigs and related information of SNP were submitted to Sequenom MassArray method for nucleic acid mass spectrometry by Beijing Conpson organism Limited.
Adding PCR amplification primer (P) with sample DNA as template1And P2) And carrying out PCR amplification reaction on the single base extension primer, purifying a reaction product, co-crystallizing the reaction product with a mass spectrum chip, detecting and parting by using a flight mass spectrometry, and detecting the SNP locus of the target gene.
2.3 data collation and analysis
1) Phenotypic data analysis
Descriptive statistical analysis is carried out on the birth measurement value of the pig and the birth measurement value of the birth (2-8 times) by using SAS9.2 statistical analysis software.
2) Haplotype analysis
The calculation of the genotype and allele frequency of the single nucleotide polymorphism was performed using PopGene 3.2. The GLM process in SAS9.2 software is used for analyzing the association analysis of SNP and litter size traits, and a fixed effect model is as follows:
y＝μ+gi+mk+e
y is a phenotypic record of reproductive traits; μ is the total average of the traits; gi is the genotype effect; mk is the production month effect; e is the random error. Data are expressed as probability values and mean ± standard deviation, with P values < 0.05 indicating significant differences.
2.4 analysis of results
The SNP typing results of HSD17B1 gene of the white pig were obtained by Sequenom MassArray as described above, and are shown in Table 1.
TABLE 1
The genotype detection result shows that: the genotype of the 5-head pig is AA genotype, the genotype of the 19-head pig is AG genotype, and the genotype of the 67-head pig is GG genotype. The results of detecting the genotype frequency of the porcine HSD17B1 gene in a swinery show that: the AA genotype frequency is 0.055, the AG genotype frequency is 0.209, the GG genotype frequency is 0.736, the GG and AG genotype frequencies are higher than those of AA, and the GG and AG genotypes are dominant genes.
And performing statistical analysis on the genotype and the reproductive performance by using SAS9.2 software, and performing multiple comparison among samples. The results are shown in Table 2.
TABLE 2
As can be seen from Table 2, the differences in litter size and average weight of embryos between different genotypes of HSD17B1 were extremely significant. The litter size of the AA type is lower than that of the AG and GG types, the initial average fetal weight of the AG type is lower than that of the AA and GG types, and the litter weight is higher than that of the AA and GG types. In the parity data, the litter size, litter weight and average weight average of the AG and GG types were higher than those of the AA type.
Therefore, the SNP (rs323407415) site obviously influences the farrowing traits of the pigs, and AG and GG genotype pigs have higher breeding efficiency in actual pig breeding.
In summary, the nucleotide at rs323407415 site of the pig HSD17B1 gene can be determined to determine whether the pig individual is of the AA genotype or the AG and GG genotypes, thereby assisting in identifying the initial/parity litter size and the average fetal weight: AG and GG genotype pigs have higher reproductive efficiency.
The AA genotype is a homozygote of the 323407415 th nucleotide of the porcine HSD17B1 gene, namely A.
The AG genotype is a heterozygote of the 323407415 th nucleotide of the porcine HSD17B1 gene, A and G;
the GG genotype is a homozygote of the 323407415 th nucleotide of the porcine HSD17B1 gene, namely G.
Example 2 detection of expression level of HSD17B1 Gene
1. Experimental sample Collection
The white pig and the Meishan pig for detecting the expression of the HSD17B1 gene are from a pig farm of the animal veterinary research institute of Beijing, China academy of agricultural sciences in Wuqing, Tianjin. Collecting ovaries of sows at 49 days of gestation, storing the samples in liquid nitrogen, and extracting total RNA. Three individuals were collected for each variety as biological replicates.
2. Detection of HSD17B1 Gene expression
2.1 Total RNA extraction from ovarian tissue
Glassware and tweezers used in the RNA extraction process need to be subjected to dry baking for 4 hours at 200 ℃ to inactivate the RNase, an Eppendorf tube and a pipette tip need to use RNase inactivation products, and experimenters need to wear masks and latex gloves in the whole experimental process.
(1) Collecting samples: placing the collected ovarian tissues in a 1.5mL Eppendorf centrifuge tube, and operating according to the RNA extraction instruction;
(2) grinding the tissue on ice by using a grinding pestle, adding 1ml of lysis solution, violently shaking for 30s, standing for 10min at room temperature, and fully lysing the tissue cells;
(3) adding 0.2mL chloroform (chloroform: Trizol volume ratio is 1:5), vigorously shaking for 15s, standing at room temperature for 10min, centrifuging at 12000g at 4 deg.C for 15min, and repeating the steps until extraction is complete;
(4) sucking the upper aqueous phase (about 450 mu L) into a new Eppendorf tube, adding isopropanol with the same volume, gently mixing, standing on ice for 20min, and centrifuging at 12000g for 10min at 4 ℃;
(5) centrifuging to obtain white colloidal precipitate at the bottom of the tube, discarding the supernatant, washing the precipitate with 1mL of 75% ethanol (prepared with DEPC water), centrifuging at 4 deg.C for 10min at 12000g, discarding ethanol, and air drying on a clean bench for 5-10min until the precipitate is semitransparent;
(6) adding DEPC water to dissolve the RNA precipitate, and determining the amount of the added DEPC water according to the amount of the precipitate;
(7) and measuring the RNA concentration of the sample by using an ultraviolet spectrophotometer.
2.2 Total RNA quality detection method
1 μ L of RNA in the extracted sample was collected, and the quality of RNA was measured by Agilent 2100Bioanalyzer and agarose gel electrophoresis. Taking a sample with the RIN value (RNA integer number) of more than 8 as a library establishing sample. And simultaneously, detecting the integrity of the RNA by adopting 2% agarose gel electrophoresis, and selecting a sample with high integrity for subsequent mRNA quantification.
2.3 reverse transcription of mRNA
Measuring RNA concentration with ultraviolet spectrophotometer, adding 2 μ g RNA into 2 μ L Oligo (dT), adding DEPC water to make up volume to 13 μ L, mixing well, water bathing at 72 deg.C for 5min, standing on ice for 3-5min, centrifuging, adding the following substances, mixing well, water bathing at 42 deg.C for 60min, and storing at-20 deg.C.
TABLE 3 reverse transcription reaction System
2.4 quantitative PCR
The Real-time quantitative PCR reaction was performed according to the kit instructions of TaKaRa SYBR Premix EX Taq (TaKaRa, DRR 041S). Each sample is provided with three replicates, the reaction is carried out in an ABI Prism 7500 real-time fluorescence quantitative PCR system, and beta-actin is an internal reference. The PCR reaction system is shown in Table 4, the reaction conditions are shown in Table 5, and the primer sequences are shown in Table 6.
TABLE 4RT-qPCR reaction System
TABLE 5RT-qPCR amplification procedure
TABLE 6mRNAqPCR primers
2.5 analysis of results
The Meishan pigs are native to the areas such as Jiading in Shanghai, Taicang in Jiangsu and the like in China, are one of the most productive and most litter-size pig breeds in China, are 30-40% higher than the litter size of modern European pig breeds such as white pigs and the like in the same litter, and are also one of the most productive pig breeds in the world so far. By comparing the HSD17B1 gene expression level of Meishan pigs with that of big white pigs, the influence of the HSD17B1 gene expression level on farrowing traits can be effectively proved. The results are shown in FIG. 1.
As can be seen from FIG. 1, the expression level of HSD17B1 in meishan pigs with high litter size is significantly lower than that in white pigs. HSD17B1 is a representative short-chain dehydroreductase in HSD17 beta family, and has very important functions in the process of estrogen synthesis. The HSD17B1 gene may influence the synthesis and metabolism of hormones, thereby affecting farrowing performance in pigs.
Example 3 kit for detecting related SNP sites
The SNP locus detection kit comprises the following components:
(1)，dNTP(2.5mM)；
(2) 5 × PCR buffer;
(3) FastPFDNA polymerase (250U);
(4) SNaPshot mix;
(5)，MgSO4solution (50 mM);
(6)，6×DNA Loading Buffer；
(7) PCR amplification primer (P)1And P2): the nucleotide sequences are shown as SEQ ID No.1 and SEQ ID No. 2;
(8) and extending a primer: the nucleotide sequence is shown as SEQ ID No. 3;
(9) exonuclease (250U);
(10) shrimp-alkaline phosphatase (100U);
(11) buffer for purification: (ii) a
(12)，ddNTP；
(13) Positive control specimen of SNP site homozygous mutation: artificially synthesizing a plasmid containing a pig rs323407415 locus into a G base;
(14) positive control specimen of SNP site heterozygous mutation: a mixture of two plasmids containing porcine rs323407415 locus artificially synthesized A and G basic groups;
(15) SNP site negative control specimen: artificially synthesized plasmid with random base.
Through the SNP site detection kit and the use method thereof, the 323407415 site SNP of the porcine HSD17B1 gene is detected.
1. DNA extraction
Appropriate amount of pig ear tissue is cut and extracted to obtain whole genome DNA.
2. PCR amplification reaction
The PCR reaction reagents (20. mu.L) included: mu.L of forward primer set (10. mu.M), 1. mu.L of reverse primer set (10. mu.M), 4. mu.L of 5 XPCR buffer, 2. mu.L of dNTP (2.5mM), 0.2. mu.L of FastPFA DNA polymerase (250U), H2O was supplemented to 20. mu.L. The PCR reaction system consists of a DNA sample to be detected (200ng), an SNP homozygous mutation positive control specimen (100ng), an SNP heterozygous mutation positive control specimen (100ng), an SNP negative control specimen (100ng) and the PCR reaction reagent.
3. PCR product identification and purification
After the PCR amplification is finished, agarose gel electrophoresis is carried out to identify the PCR product. Purifying the PCR amplification product by using shrimp-alkaline phosphatase and exonuclease, carrying out enzyme digestion at 37 ℃ for 60min, and inactivating the shrimp-alkaline phosphatase and the exonuclease at 80 ℃ for 15 min. The purification system comprises: mu.L of shrimp-alkaline phosphatase (0.6U) at the final concentration, 5. mu.L of exonuclease (1.2U) at the final concentration, 50ng of PCR amplification product, and 2. mu.L of buffer for purification.
4. Extension reaction
The amplification product was recovered, followed by PCR amplification using the extension primer in a system containing 0.1mM ddNTP.
5. Capillary electrophoresis and SNP site analysis
The extended primers were sequenced using an ABI3730XL sequencer. The determination of deoxynucleotides at SNP sites was carried out on the resulting peak maps using GeneMapper series software.
The invention has been described in detail with reference to specific embodiments and illustrative examples, but the description is not intended to be construed in a limiting sense. Those skilled in the art will appreciate that various equivalent substitutions, modifications or improvements may be made to the technical solution of the present invention and its embodiments without departing from the spirit and scope of the present invention, which fall within the scope of the present invention. The scope of the invention is defined by the appended claims.
Sequence listing
<110> Beijing animal husbandry and veterinary institute of Chinese academy of agricultural sciences
<120> swine polyculture integration accurate breeding method
<130> 2010
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 29
<212> DNA
<213> porcine HSD17B1 gene SNP site amplification primer (Sus scrofa)
<400> 1
acgttggatg acgggcttcg cttgcttct 29
<210> 2
<211> 30
<212> DNA
<213> porcine HSD17B1 gene SNP site amplification primer (Sus scrofa)
<400> 2
acgttggatg aaacagcgtt gaagggaagc 30
<210> 3
<211> 15
<212> DNA
<213> porcine HSD17B1 gene SNP site extension primer (Sus scrofa)
<400> 3
cttgcttctg ctgct 15
<210> 4
<211> 20
<212> DNA
<213> porcine HSD17B1 gene mRNA expression level PCR amplification primer (Sus scrofa)
<400> 4
tccaatggag aagatggcgg 20
<210> 5
<211> 20
<212> DNA
<213> porcine HSD17B1 gene mRNA expression level PCR amplification primer (Sus scrofa)
<400> 5
ggagatgtcc ggttcagagc 20
Claims (5)
1. A breeding method of high-yield litter size/weight pigs, which comprises the step of selecting pigs with GG or AG genotype at position 323407415 of HSD17B1 gene as parents for hybridization;
compared with the pigs with the genotypes of AA, the pigs with the 323407415 locus of the HSD17B1 gene have higher litter size and litter weight,
the method for detecting 323407415 th site of HSD17B1 gene comprises a group of amplification primers P1And P2The amplification primer P1And P2The nucleotide sequences of (A) are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2;
the method for detecting the 323407415 locus of the HSD17B1 gene further comprises an extension primer, wherein the nucleotide sequence of the extension primer is shown as a sequence in SEQ ID NO. 3;
the pig is white pig or Meishan pig.
2. A breeding method according to claim 1, characterized in that the 323407415 genotype of HSD17B1 gene is detected by using a kit, wherein the kit comprises PCR reaction reagent, PCR amplification primer, PCR product purification reagent, extension primer and SNP site control sample, and the nucleotide sequences of the amplification primer are respectively shown as SEQ ID No.1 and SEQ ID No. 2;
the nucleotide sequence of the extension primer is shown as a sequence shown in SEQ ID NO. 3.
3. A breeding method according to claim 1, characterized in that the breeding method comprises the steps of:
step 1, selecting a pig with the 323407415 th site of HSD17B1 gene being GG or AG genotype as a parent to hybridize to obtain an F1 generation piglet;
step 2, carrying out genotype analysis on the F1 generation piglets, breeding piglets with GG or AG genotypes at the 323407415 th site of the HSD17B1 gene, and continuously hybridizing after the piglets grow up to obtain F2 generation piglets;
step 3, carrying out genotype analysis on the F2 generation piglets, breeding piglets with GG or AG genotypes at the 323407415 th site of the HSD17B1 gene, and continuously hybridizing after the piglets grow up to obtain F3 generation piglets;
and (3) repeating the step 2 or the step 3 to finally obtain the breeding pig with the genotype GG high inheritance.
4. The application of the reagent for detecting the genotype of 323407415 locus of HSD17B1 gene in identification or auxiliary identification of pig farrowing traits and breeding is characterized in that the pigs with the genotypes of AG and GG at 323407415 locus of HSD17B1 gene have higher litter size and litter weight compared with the pigs with the genotypes of AA, and the pigs are white pigs and Meishan pigs.
5. The use according to claim 4, wherein the reagent comprises an amplification primer and an extension primer, the nucleotide sequences of the amplification primer are shown as SEQ ID No.1 and SEQ ID No.2, respectively, and the nucleotide sequence of the extension primer is shown as SEQ ID No. 3.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996033288A1 (en) * | 1995-04-17 | 1996-10-24 | Dekalb Swine Breeders, Inc. | Gene marker associated with swine proliferacy |
| CN104651356A (en) * | 2015-03-16 | 2015-05-27 | 南京农业大学 | SNP (Single Nucleotide Polymorphism) marker related to farrowing traits of Erhualian sows and detection method and application of SNP marker |
| CN105483259A (en) * | 2016-01-04 | 2016-04-13 | 中国科学院昆明动物研究所 | SNP locus related to litter size on pig chromosome 12 |
| CN106011245A (en) * | 2016-06-01 | 2016-10-12 | 中国农业科学院北京畜牧兽医研究所 | Molecular marker identification method related to pig birth litter weight and fetal weight and application of molecular marker identification method |
-
2017
- 2017-09-11 CN CN201710813414.XA patent/CN109486956B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996033288A1 (en) * | 1995-04-17 | 1996-10-24 | Dekalb Swine Breeders, Inc. | Gene marker associated with swine proliferacy |
| CN104651356A (en) * | 2015-03-16 | 2015-05-27 | 南京农业大学 | SNP (Single Nucleotide Polymorphism) marker related to farrowing traits of Erhualian sows and detection method and application of SNP marker |
| CN105483259A (en) * | 2016-01-04 | 2016-04-13 | 中国科学院昆明动物研究所 | SNP locus related to litter size on pig chromosome 12 |
| CN106011245A (en) * | 2016-06-01 | 2016-10-12 | 中国农业科学院北京畜牧兽医研究所 | Molecular marker identification method related to pig birth litter weight and fetal weight and application of molecular marker identification method |
Non-Patent Citations (5)
| Title |
|---|
| Association of CYP19A1 gene polymorphisms with reproductive traits in pigs;ZHOU R et al.;《Journal of Integrative Agriculture》;20170731;第16卷(第7期);第1558–1565页 * |
| Polymorphism of the pig 17beta-hydroxysteroid dehydrogenase type1 (HSD17B1) gene and its association with reproductive traits;Liu LQ et al.;《Animal Reproduction Science》;20080912;第114卷(第1-3期);摘要，材料和方法，结果和讨论，图1，表2 * |
| rs323407415;WU_ABGC;《USUC dbSNP》;20140128;同上 * |
| WU_ABGC.rs323407415.《USUC dbSNP》.2014,rs323407415和SNP侧翼序列比对的基因组序列. * |
| 猪重要性状全基因组关联分析的研究进展;赵谦等;《畜牧兽医学报》;20150615;第46卷(第6期);第873-881页 * |
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